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- PDB-6pkl: MicroED structure of proteinase K from an uncoated, single lamell... -

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Basic information

Entry
Database: PDB / ID: 6pkl
TitleMicroED structure of proteinase K from an uncoated, single lamella at 2.59A resolution (#7)
ComponentsProteinase K
KeywordsHYDROLASE
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding
Similarity search - Function
Proteinase K-like catalytic domain / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / : / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site ...Proteinase K-like catalytic domain / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / : / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.59 Å
AuthorsMartynowycz, M.W. / Zhao, W. / Hattne, J. / Jensen, G.J. / Gonen, T.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35 GM122588 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2P50GM082545 United States
CitationJournal: Structure / Year: 2019
Title: Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED.
Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction ...Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.
History
DepositionJun 29, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Proteinase K


Theoretical massNumber of molelcules
Total (without water)28,9311
Polymers28,9311
Non-polymers00
Water41423
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: PISA
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area9940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.500, 67.500, 105.210
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Proteinase K / Endopeptidase K / Tritirachium alkaline proteinase


Mass: 28930.783 Da / Num. of mol.: 1 / Fragment: UNP residues 106-384 / Source method: isolated from a natural source / Source: (natural) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 23 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Proteinase K / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.02893 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 7.5
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Proteinase K purchased from Sigma
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 273 K

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 3 sec. / Electron dose: 0.03 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 100 / Num. of grids imaged: 1 / Num. of real images: 100
Details: Continuous rotation with a rotation rate of 0.2 degrees per second and a readout every 3 seconds
Image scansSampling size: 28 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 2055 mm
EM diffraction shellResolution: 2.59→43.47 Å / Fourier space coverage: 92.89 % / Multiplicity: 4.7 / Num. of structure factors: 3727 / Phase residual: 28.73 °
EM diffraction statsFourier space coverage: 92.89 % / High resolution: 2.59 Å / Num. of intensities measured: 34844 / Num. of structure factors: 3727 / Phase error: 28.73 ° / Phase residual: 28.73 ° / Phase error rejection criteria: 0 / Rmerge: 0.53 / Rsym: 0.6

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Processing

SoftwareName: PHENIX / Version: (1.14_3260: ???) / Classification: refinement
EM software
IDNameVersionCategory
6PHENIX1.15model fitting
8PHENIX2.8.2molecular replacement
12PHENIX1.15.23D reconstruction
13PHENIX1.15model refinement
Image processingDetails: This was the new CetaD.
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.5 Å / B: 67.5 Å / C: 105.2 Å / Space group name: 96 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 2.59 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 20.7 / Protocol: OTHER / Space: RECIPROCAL
Atomic model buildingPDB-ID: 6CL7

6cl7


Pdb chain-ID: A / Pdb chain residue range: 106-384
RefinementResolution: 2.59→43.466 Å / SU ML: 0.36 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 28.81
RfactorNum. reflection% reflectionSelection details
Rfree0.3102 868 6.47 %copied from 6cl7
Rwork0.256 ---
obs0.2597 7475 92.89 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0072070
ELECTRON CRYSTALLOGRAPHYf_angle_d0.6512814
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d3.2181208
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.042312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.003370
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5901-2.75230.34341470.26552124ELECTRON CRYSTALLOGRAPHY95
2.7523-2.96480.37371430.30662140ELECTRON CRYSTALLOGRAPHY94
2.9648-3.26310.34131420.2962108ELECTRON CRYSTALLOGRAPHY94
3.2631-3.7350.33941410.26232108ELECTRON CRYSTALLOGRAPHY93
3.735-4.70480.25721450.2162067ELECTRON CRYSTALLOGRAPHY92
4.7048-43.47210.27351500.23482010ELECTRON CRYSTALLOGRAPHY89

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